Spinal interneuron transplantation to repair the injured cervical spinal cord

Cervical spinal cord injuries directly compromise the phrenic motor network resulting in diaphragm paralysis and impaired breathing. Anatomically, injuries at this level result in extensive cell loss including phrenic interneurons and phrenic motorneurons. Despite these consequences, some limited plasticity typically occurs weeks post-injury shown by increased interneuronal integration with the damaged phrenic network and modest improvement of diaphragm recovery. In an effort to harness and enhance this plasticity, we have engineered stem-cell derived spinal interneurons that can be transplanted directly into the injured spinal cord. We recently found that transplanting neural precursor cells (NPCs) comprised of spinal neural precursors promotes phrenic recovery post-SCI. Building on these initial results, the present dissertation tests the hypothesis that enriching these neural precursors with stem cell derived V2a and V0 spinal interneurons (SpIN), will further enhance diaphragm recovery. Adult, female, Sprague Dawley rats received SpIN-enriched NPC transplants sub-acutely (1 week) following a lateralized, mid-cervical contusion injury. Donor SpINs were engineered to express the hM4Di receptor, allowing for inhibition of these cells with application of clozapine. Multi-electrode array recordings were used to confirm inhibition of donor SpINs in vitro prior to transplantation. Intraspinal clozapine application during diaphragm electromyography was used to silence transplanted V2a SpINs, resulting in decreased diaphragm output following drug delivery. Pseudorabies virus tracing of the injured phrenic network was used to show synaptic integration of donor NPCs and V2a and lack of connectivity of V0 SpINs with host phrenic circuit. Ipsilateral diaphragm activity was unaffected by transplantation of NPC enriched with V0 SpINs while there was an increase in the contralateral diaphragm output. Future studies will look at labeling connectivity from the contralateral hemidiaphragm, and further RNAscope experiments to quantify anatomical connections made by donor SpINs. This research provides the first in depth assessment of how these donor SpIN populations contribute to phrenic recovery.